The purpose of this investigation is to study the synthesis and properties of an onco-developmental protein produced by tumor cells growing in cell culture. To assomplish this goal the synthesis rates, molecular variants and properties of rat alpha-fetoprotein (AFP) produced by mass and clonal cultures of cell derived from chemically-induced hepatocellular carcinomas will be studied. AFP synthesis by the cells in culture will be compared to AFP synthesis in vivo by normal fetal/newborn animals, by the hepatocellular carcinomas from which the cultured cells have been derived and by tumors which arise following injection of cultured cells into syngeneic rats. Correlations between tumorigenicity and AFP synthesis will be sought. Hepatocellular carcinoma-derived cells are grown using methods which have been modified to support the growth of epithelial cells. Cell lines have been established from Morris hepatoma 7777 and transplantable hepatomas F33 and F41. Six clonal cell lines have been established from the mass culture of hepatoma 7777. The relationship of these cells to liver parenchymal cells will be established by cell morphology, albumin synthesis, hydrocortisone-inducible tyrosine aminotransferase activity and the transformation of ornithine to arginine. The synthesis rates, processing, secretion and molecular variants of AFP produced by the cultured cells will be characterized. The effect of culture conditions, hormones, cyclic nucleotides and carcinogens on AFP production in vitro will be tested. The relationship of cell cycle to AFP secretion will be examined using parasynchronous cell cultures. Correlations between AFP secretion and growth properties of the cells will be examined. AFP standards will be prepared by immunoadsorbent affinity chromatography. AFP concentrations will be measured immunochemically. Concanavalin A-affinity molecular variants of AFP will be analyzed by concanavalin A-affinity chromatography. Electrophoretic molecular variants of AFP will be analyzed by isoelectric focusing and two-dimensional single electroimmunodiffusion. The molecular size of AFP produced in vitro will be determined by gel filtration chromatography.